Study On The Isolation And Purification, MPEG Modification And Stability Of Chicken Egg Yolk Immunoglobulin

Chicken egg yolk of immunoglobulin, short for IgY, also known as chicken egg yolk antibody, was an immunoglobulin in the egg yolk. It was produced by laying hens which were immuned. During the immune response in the fallopian tube, IgG in blood was selectivetransferred to yolk. As IgY is protein with large molecular weight and a certain degree of immunogenicity, it can lead to produce antibodies to reduce the effect when used for the body. Inaddition, IgY is sensitive to temperature, pH and enzyme thus the application of IgY was limited. To solve this problem, IgY was modified with mPEG, and the main cotents which were studied as follows:chicken IgY separation and purification, mPEG chemical modification and the stability of IgY and modified production.1. An economical, effective and large-scale method for the preparation of immunoglobulin of chicken egg yolk (IgY) was developed. The method was based on an improved water dilution combining with PEG-6000precipitation and anion-exchange chromatography. The specified processes were as follows:egg yolk was obtained from fresh eggs separation, water dilution, the supernatant was got after centrifuge. Then the crude IgY was obtained from the supernation with PEG-6000precipitation followed by dialysis and freeze-dried. After one step anion exchange chromatography, the pure IgY was obtained. The experimental results showed that, egg yolk was diluted with8volumes of aseptic water, adjusted the pH to5.2with0.1mol/L HCl, then the mixture was centrifuged at5000X g after standing for8h at4℃. The crude IgY was obtained from the supernation with the yields of93.47%. The purification of IgY was achieved by6%PEG-6000precipitation and DEAE-Toyopearl650M anion-exchange chromatographic packing. The optimum purified conditions by ion-exchange chromatography were as follows:equilibrated with0.05mol/L phosphate buffer (pH7.0) and eluted with0.075mol/L phosphate buffer (pH7.0). The purity of the IgY fraction was consistently greater than95%with high activity (73.77%) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This method was superior to the traditional ion-exchange chromatography that can’t simultaneously obtain high purity and high recovery. Moreover, this method is suitable for a large-scale IgY preparation from chicken egg yolk.2. The effect of monomethoxypolyethlene glycol (mPEG) modification on the stability of chicken IgY and compare the stability of the modification products were studied by Fourier Transform Infrared spectroscopy (FT-IR), CD spectrooscopy and Fluorescence spectroscopy. The specified processes were as follows:The mPEG was activated by NHS, got NHS-mPEG, frozen dry, chemistry modification of IgY by NHS-mPEG, then mPEG-IgY was obtained. NHS-mPEG was used to modify IgY after mPEG activated with N-hydroxysuccinimide (NHS). The optimal reaction condition for modification was1:10molar rate of IgY to mPEG at pH7, reaction for1h, the product was obtained with modification rate of20.56%and activity reservation of87.62%. The spectrooscopy results showed that the a-Helix, P-sheet, P-Turn, Random content of IgY was14.5%,42.1%,6.2%,37.2%, while mPEG-IgY was9.1%,44.9%,7.4%,38.6%respectively. The comparison showed that, a-Helix content of mPEG-IgY decreased by about37%, β-sheet content increased and other content did not change significantly.3. The thermal stability of IgY and mPEG-IgY was compared by Spectroscopic methods. The results showed that the a-Helix, P-sheet, P-Turn, Random content of IgY changed from14.5%,42.1%,6.2%,37.2%to1.6%,55.25%,5.8%,37.5%, while mPEG changed from12.9%,42.7%,6.3%,38.1%to3.1%,50.5%,7.2%,39.2%, respectively, after incubating120min at70℃. The thermodynamic analysis showed that, the class of IgY ang mPEG-IgY thermal denaturation was1. Under the heating conditions of65,70,75,80,85,90℃, thermodynamics parameters for IgY were as follows:D value:208.33,60.61,8.02,4.48,2.50,2.18min, Z value:11.5℃, the apparent activation energy(Ea):200.841kJ/mol, the enthalpy value:198.0296、197.988、197.9465、197.9049、197.8633、197.8218kJ/mol, entropy value:58.6675、58.65283、58.63837、58.6241、58.59619kJ/(mol·K), Gibbs free energy:33.09298、30.65444、28.2166、25.77931、23.34261、20.90646kJ/mol, respectively. While for mPEG-IgY, D value:208.33,60.61,8.02,4.48,2.50,2.18min, Z value:12.22℃, the apparent activation energy(Ea):186.915kJ/mol, the enthalpy value:84.1036,184.0621 184.0205、183.9789、183.9373、183.5958kJ/mol, entropy value:53.50651、53.49183、53.47737、53.62411、58.61005、58.59619kJ/(mol·K), Gibbs free energy:33.67654、31.45257、29.2292、27.00644、24.78425、22.26271kJ/mol, respectively.4. The pH stability of IgY and mPEG-IgY was also studied. The results showed that, activity retention of mPEG-IgY was nearly five times higher than IgY at pH4and pH11. The circular dichroism spectrum analysis showed that, for the treatment with acid at pH4, a-Helix content of IgY decreased by76.4%, while it’s lower by15.7%for mPEG-IgY. The content of β-sheet were both increased with18.4%and29.7%for IgY and mPEG-IgY, respectively. For the treatment with base at pH11, a-Helix content of IgY decreased by71.1%, while it’s lower by14.9%for mPEG-IgY. The content of β-sheet were both increased with16.5%and21.7%for IgY and mPEG-IgY, respectively. Thus, it indicated that, IgY modified with mPEG had a higher tolerance to pH.5. The stability of IgY and mPEG-IgY in the digestive systerm of artificial simulated gastrointestinal was studied. During the simulated gasttic digestion at pH4, it could be found that the activity retention of IgY changed from83.57%to55.59%with a reduction of28.08%in the30-45min period of the reaction. While for mPEG-IgY, the activity retention of IgY changed from89.81%to72.63%with a reduction of17.18%. In the90-120min period of the reaction, the activity retention of IgY changed from46.24%to31.33%with a reduction of14.91%. While for mPEG-IgY, the activity retention of mPEG-IgY changed from63.21%to60.44%with a reduction of2.77%. During the simulated gasttic digestion at pH2, the activity retention of IgY changed from71.69%to50.37%with a reduction of21.32%in the30-45min period of the reaction. While for mPEG-IgY, the activity retention of IgY changed from83.33%to65.35%with a reduction of18.08%. In the90-120min period of the reaction, the activity retention of IgY changed from41.36%to20.69%with a reduction of20.67%. While for mPEG-IgY, the activity retention of mPEG-IgY changed from55.49%to49.82%with a reduction of5.67%. The amino acid composition analysis showed that, the amino acid compositon content of IgY was basically the same with mPEG-IgY, indicating that the primary structure of IgY was not destroyed after modification with mPEG. The oligopeptide component analysis showed that, the type and quantity of mPEG-IgY reduced after treating with digestive juice. It indicating that IgYmodified with mPEG hold more resilience and the activity was also to be maintained. Therefor, we can draw the conclusion that IgY modified with mPEG had a higher stability against pepsin and trypsin.